The present invention relates to genes encoding the type B subunit protein of avian lactate dehydrogenase.
Lactate dehydrogenase (LDH) is an enzyme which dehydrogenates lactic acid into pyruvic acid in conjunction with the hydrogen acceptor NAD.sup.+ and which exists widely in a variety of animal tissues and microorganisms as an enzyme serving to produce lactic acid from pyruvic acid in the glycolytic pathway.
Particularly, it is known that animal LDH is grouped into two subunits, type A (type M or skeltal muscle type) and type B (type H or heart muscle type), which results in a variation of enzymological properties between LDH isozymes. The subunits homogeneously or heterogeneously form tetrameric isozymes called as LDH.sub.1 (B.sub.4), LDH.sub.2 (A.sub.1 B.sub.3), LDH.sub.3 (A.sub.2 B.sub.2), LDH.sub.4 (A.sub.3 B.sub.1), LDH.sub.5 (A.sub.4) in descending order of electric mobility toward the anode side. Distributions of each isozyme in a body are uneven, e.g. LDH-B.sub.4 is found at higher level in heart muscle, red blood cells and kidney while LDH-A.sub.4 is found at higher level in skeltal muscle and liver. LDH-A.sub.2 B.sub.2 is found at higher level in prostate gland, colon, thyroid and lung tissues. In recent years, the presence of types C and D subunits has newly been identified, and LDH-X(C.sub.4) has been detected in testis and sperms as well as LDH-Z(D.sub.4) in choriocarcinoma and metastasis lesions thereof.
LDH has been used especially in the field of clinical test reagents 1) as a coupling enzyme for determining the enzymatic activity of various amino-transferases such as alanine aminotransferase (ALT) in UV spectrometry of the produced pyruvic acid, 2) as a coupling enzyme converting various substrates such as urea into pyruvic acid in UV spectrometry of the produced pyruvic acid, and 3) for eliminating endogenous pyruvic acid in a subject. Particularly, the determination of aminotransferase activity using LDH as mentioned in 1) is widely adopted as a clinical test, because amino-transferases are enzymes which show high activity in liver, heart, kidney, etc. and remarkably increase in serum during various diseases.
It is preferable for clinical test reagents to be stable in a liquid state rather than a powder state in view of convenience. Porcine LDH-B.sub.4 has been commonly used as a clinical test reagent for determining amino-transferase activity because it is abundantly available, but it was disadvantageously difficult to store stably in a liquid state for a long period.
Recently, LDH-B.sub.4 derived from birds has been found to show remarkable heat stability in solution even in a pH range ensuring the stability of the coenzyme (NADH), such as a pH of about 9 to 10. The LDH-B.sub.4 derived from birds has the almost same enzymatic properties as the prior porcine LDH-B.sub.4 and does not cause any special problem in the determination sensitivity when it is used as a reagent for determining transaminase activity. Therefore, the LDH-B.sub.4 derived from birds is expected to be sufficiently useful as an enzyme for clinical tests from its stability in solution and enzymatic properties (Japanese Patent Public Disclosure: No. 289/1996 "Transaminase Determination Reagent"; and "Heat Resistant Lactate Dehydrogenase Appropriate to Liquid ALT Determination Reagent", Clinical Chemistry, Supplement, No. 2 to Vol. 23,, 1994, 141b).
However, it is difficult to practically use the natural LDH-B.sub.4 protein prepared from birds as a clinical test enzyme because it can be obtained only in a small amount. Thus, it is important to clone a LDH-B gene derived from birds so that a large amount of LDH-B.sub.4 protein may be expressed by genetic engineering techniques.